Determination of Allicin in Allium Sativum Using High Performance Liquid Chromatography and Study of Genotoxic Effect on Human Leukocytes

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Vol 8, Issue 6, 2015 ISSN - 0974-2441 Research Article DETERMINATION OF ALLICIN IN ALLIUM SATIVUM USING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY AND STUDY OF GENOTOXIC EFFECT ON HUMAN LEUKOCYTES

NISHU SEKAR, RAJIV SUNDARAMOORTHY, SRISTI MAJUMDAR, KRISHNAKSHI BHUYAN, JYOTIREKHA DAS, ABILASH VALSALA GOPALAKRISHNAN* Division of Biomolecules and Genetics, School of Biosciences and Technology, VIT University, Vellore, Tamil Nadu, India. Email: [email protected] Received: 21 July 2015, Revised and Accepted: 07 September 2015

ABSTRACT

Objectives: Allicin is an organosulfur compound featuring thiosulfinate functional group. The compound is formed in garlic after tissue damage, by the action of enzyme alliinase on alliin. This study involves extraction of allicin from garlic using high-performance liquid chromatography (HPLC) and study of it is the genotoxic effect on human leukocytes.

Methods: A simple and rapid reverse phase HPLC was used for the extraction of allicin. Quality allicin, in conjugation with chymosin, was used to study its genotoxic effect on leukocytes.

Results: Garlic oil and garlic extract showed similar retention time, and we measured the products using genotoxic effects in human leukocyte culture and it shows statistically not significant.

Conclusion: This study suggested that to take a lower concentration of garlic extracts benefits for health and these findings useful for further research.

Keywords: Allium sativum, Leukocytes, Chromosome, Genotoxic, High-performance liquid chromatography.

INTRODUCTION METHODS

Garlic (Allium sativum) possesses exquisite defense system and Allicin extraction several therapeutical properties, contributed by several sulfur- The outer skin of the garlic cloves was peeled and crushed in a garlic containing compounds, mainly allicin. Allicin (C3H5SS(O)C3H5) press. The pressed garlic was then collected in a beaker and mixed is an organosulfur compound, first isolated and studied in the thoroughly. 700-900 mg of the pressed mash was weighed and laboratory [1]. It is known as 2-propene-1-sulfinothioic acid S-2- transferred to a 50 ml centrifuge tube. Using a volumetric pipette, 25 ml propenyl ester, thio-2-propene-1-sulfinic acid S-allyl ester, diellyl of cold water was delivered to the sample and immediately capped and disulfide-oxide, diallyl thiosulfinate, and percent composition: shaken vigorously for 30 seconds. Heat transfer was avoided from C - 44.4%, H - 6.21%, O - 9.86%, and S - 39.52% [2]. The interaction hands by holding the tube cap while shaking. An additional 25 ml of of non-protein amino acid alliin with the enzyme alliinase produces cold water was added and shaken for 30 more seconds to dilute and mix allicin [3]. Alliin (S-allylcysteine sulfoxide, percent composition: the solution. Each sample is filtered through 0.45 µm glass filter into C - 40.66%, H - 6.26%, N - 7.90%, O - 27.08% and S - 18.09%) HPLC vial and capped for injection. consists of a sulfoxide group, allyl group and the amino acid cysteine (contains SH rather than S=O). Alliin is synthesized from S-Allyl HPLC conditions cysteine (deoxyalliin) [4]. It possesses a wide variety of medicinal Column: Phenomex ProdigyTM ODS (3), 5 µm, 100 Å, 4.6 mm × 250 mm, properties including antimicrobial, bacteriostatic, anti-thrombotic, mobile phase:methanol:water (50:50), flow rate: 1.0 ml/minutes, anti-fungal, anti-inflammatory, anti-cancer, and anti-atherosclerotic detector: 240 nm, injection Vol.: 25 µl, column temperature: 28°C, run activities along with the capacity to lower serum lipid levels and time: 16 minutes. ocular pressure [5-8]. On comminuting fresh garlic cloves, alliin is converted into diallyl thiosulfinate (allicin) in seconds. Allicin and Procedure the other thiosulfinates are unstable, but its stability can be greatly Garlic Pearls, a soft gel of Ranbaxy, was taken as the standard. An improvedby dissolving and diluting in water [9]. extraction solvent blank of water/methanol was prepared. A single injection was made of the blank. A single injection of the reference High-performance liquid chromatography (HPLC) has superior standard was also made. A linearity curve for allicin was prepared, resolving power due to the high-pressure separation condition, with the origin ignored, using peak versus concentration (µg/ml). making it the most preferred chromatographic technique for the Linear regression analysis on the data was performed. The coefficient separation of biological compounds. For the current study, the of determination, r2 concentration of allicin in the isolated fraction is standardized prepared was made. The percent allicin in the samples against allicin by HPLC analysis. Sample analysis is carried out by extraction of reference was calculated must [10]. be ≥0.999. A single injection of the sample garlic cloves with water and quantified against the isolated allicin external standard. Many researches are being carried out to improve Leukocyte culture method the current scenario of cancer. However, until now less or no study Chromosome preparations were obtained from phytohemagglutinin has been done to check the genotoxic effect of allicin. This current (PHA)-stimulated peripheral blood lymphocytes following the modified work is an attempt to examine the effect of allicin on human blood method of Hungerford [11]. About 2 ml of venous blood sample was leukocyte. collected in a sterile heparinized syringe. 0.5 ml of the blood was Nishu et al. Asian J Pharm Clin Res, Vol 8, Issue 6, 2015, 153-156

Fig. 1: High-performance liquid chromatography analysis of garlic pearls showing the retention times

Fig. 2: High-performance liquid chromatography analysis of garlic extract showing the retention times inoculated into the vials containing 5 ml of RPMI 1640 medium containing 37°C after which it was centrifuged for 6 minutes. The supernatant 1 ml of FB serum and 0.2 ml of PHA under aseptic condition. The culture was carefully removed, and the cells were fixed with 6 ml of filtered vials were then placed in an incubator at 37°C. The cultures were shaken carnoys fixative (3:1 methanol: acetic acid). The tubes were left at room periodically, and carbon dioxide was released once every day. temperature for 2 hrs. One change of fixative was given prior to slide preparation. Treatment with standard garlic pearls At the 71st hrs of incubation, the cultures were treated with different Slide preparation concentration of standard Garlic Pearls (10, 20, 40, 80 µg/ml) for The cell pellet was suspended in a small quantity of freshly prepared 1 hrs. After 1 hrs of incubation, the culture was thoroughly washed by fixative. A test slide was prepared by gently placing a drop of the cell centrifuging the content at 1000 rpm for 10 minutes, and supernatant suspension on a cleaned glass slide and dried immediately on a hot were discarded carefully and 6 ml of fresh RPMI 1640 medium was plate (40°C). The test slide was examined under the microscope for added to the pellet and mixed well with Pasteur pipette. cell density and metaphase spreads. Other slides were prepared after suitable modifications. Treatment with extracted allicin st At the 71 hrs of incubation, the cultures were treated with different Staining procedure concentration of extracted allicin (10, 20, 40, 80 µg/ml) for 1 hrs. After The slides were stained in 4% Giemsa solution for 4 minutes and 1 hrs incubation, the culture was thoroughly washed by centrifuging the washed in distilled water for 1 minutes and then air-dried. content at 1000 rpm for 10 minutes, and supernatant were discarded carefully and 6 ml of fresh RPMI 1640 medium was added to the pellet Microscopic analysis and mixed well with Pasteur pipette. Scoring of well-spread and stained cells was performed under oil immersion (×100) of the light microscope (Leica ATC 2000). The well Culture harvesting spread metaphases were photographed using Olympus microscope After washing process, the dividing cells were arrested at metaphase by with the camera. adding two drops of 0.001% colchicine solution to each culture vial. The cultures were incubated further for 20 minutes at 37°C. The contents of RESULT AND DISCUSSION the vials were then transferred to 15 ml centrifuge tubes and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and the The chromosomal aberration analysis is one of the widely used cells were resuspended in a small amount of solution left behind by parameters for testing the protective effects of natural compounds gently tapping the tube. 6 ml of pre-warmed (37°C) hypotonic solution on the drug and chemical induced toxicity. The modulatory effect of (0.075M KCl) was added to the tubes, and the contents were mixed natural compounds on the chromosomal aberration induced by various gently using a Pasteur pipette. It was then incubated for 5 minutes at kinds of chemicals and drugs are well established [12-15].

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HPLC technique was applied for the present research work. In 2012 although the proportion and amount of various constituents may differ Rahman et al. [9] reported that except HPLC none other technique is significantly [17,18]. Allicin liquid forms thiosulfinate compounds. reliable for the study of allicin. A study showed that the retention time These components are difficult to measure or detect without HPLC. of allicin seemed to vary from 3 to 3.7 minutes/ml in several garlic Hence, in this study we used high HPLC for determination of allicin in varieties [16]. Optimum conditions were applied for extraction of garlic. This study seems to be the one of the best methods to measure allicin from Indian garlic, and garlic oil was used as a standard. HPLC genotoxic effects in human leukocytes. result showed that the retention time of standard garlic pearls is 3 minutes/ml and that of garlic extract was noted to be 2.2 minutes/ CONCLUSION ml (Fig. 1). The garlic extract had also shown similar retention time Although the analysis of allicin is difficult due to its instability, the essentially depicting the presence of allicin at a higher level in the extract current study was an attempt to determine the presence of allicin (Fig. 2). Although the analysis of allicin is difficult due to its instability, and the current study was an attempt to determine the presence of allicin in garlic extract and to check its genotoxic effect on human blood leukocytes. The methanol-garlic extract and standard were applied to the metaphase state of in vitro cultured human blood leukocyte and the chromosomal aberrations were found to be not statistically significant.

Allicin is widely used as a therapeutic agent for cancer, but no work has been done on its impact on normal blood leukocyte culture. The current study shows many chromatid and chromosomal aberrations in treated metaphase spread (Fig. 3). Hence, there is no significant difference observed between control, extract and standard samples in the following frequency: The percentage of chromosomal aberrations a b c were 1%, 5%, 6%, respectively, (Tables 1-3 and Fig. 4). Fig. 3: The representative images of metaphase chromosomes In the market, a lot of garlic based products are available. They can control and treated with allicin, (a) control, (b) chromosome be usually oil, powder or extract form. As compared to other garlic breaks, (c) chromosome deletion preparations, dehydrated garlic powder is thought to retain the same ingredients as raw garlic, both water-soluble and organic soluble,

Table 1: The chromosome aberration in control sample

Sample Number Chromosome Total number code of cells aberration of aberration scored Breaks N (%) Control 01 50 1 1 (2) Control 02 50 0 0 (2) Control 03 50 1 1 (2) Control 04 50 0 0 (0) Total 200 2 2 (1) Mean±SD 0.50±0.58 Fig. 4: Different types of chromosomal aberrations compared SD: Standard deviation with control, garlic pearls and extracted allicin

Table 2: The chromosome aberration in treated with extracted garlic product

Concentration Number of Chromatid break Chromosome aberration Total number of aberration cells scored Breaks Deletion Breaks N (%) 10 µl 50 1 0 0 1 (2) 20 µl 50 0 0 1 1 (2) 40 µl 50 2 0 1 3 (6) 80 µl 50 1 3 1 5 (10) Total 200 4 3 3 10 (5) Mean±SD 1±0.82 0.75±1.50 0.75±0.50 SD: Standard deviation

Table 3: The chromosome aberration in treated with standard garlic pearls

Concentration Number of Chromatid break Chromosome aberration Total number of aberration cells scored Breaks Deletion Breaks N (%) 10 µl 50 1 0 0 1 (2) 20 µl 50 1 0 0 1 (2) 40 µl 50 2 0 2 4 (8) 80 µl 50 3 2 1 6 (12) Total 200 7 2 3 12 (6) Mean±SD 1.75±0.96 0.75±0.96 0.50±1 SD: Standard deviation

155 Nishu et al. Asian J Pharm Clin Res, Vol 8, Issue 6, 2015, 153-156 in garlic extract and to check its genotoxic effect on human blood 6. Block E. The organosulfur chemistry of the genus Allium - Implications leukocytes. Rahman et al. [9] reported that except HPLC none other for the organic chemistry of sulphur. Angew Chem 1992;31(9):1135-78. technique is reliable for the study of allicin. In reference to different 7. Koch HP, Lawson LD. Garlic: The Science and Therapeutic Application nd literatures, HPLC technique was applied for the present research of Allium sativum L. and Related Species. 2 ed. Baltimore: Williams work. From the HPLC result, we conclude that the garlic oil used as a and Wilkins; 1996. p. 329. 8. Lawson LD. Garlic: A review of its medicinal effects and indicated standard for allicin showed a retention time of 3 minutes/ml. The garlic active compounds. In: Phytomedicines of Europe: Their Chemistry extract had also shown similar retention time essentially depicting the and Biological Activity. Washington, DC: American Chemical Society; presence of allicin in a higher level in the extract. The methanol-garlic 1998. p. 176-209. extract and standard were applied on the metaphase state of in vitro 9. Rahman MM, Fazlic V, Saad NW. Antioxidant properties of raw garlic cultured human blood leukocyte, and the chromosomal aberrations (Allium sativam) extract. Int Food Res J 2012;19(2):589-91. were found to be statistically not significant. 10. Lawson LD, Wood SG, Hughes BG. HPLC analysis of allicin and other thiosulfinates in garlic clove homogenates. Planta Med ACKNOWLEDGMENT 1991;57(3):263-70. 11. Hungerford DA. Leukocytes cultured from small inocula of whole The authors would like to express their gratitude to VIT University blood and the preparation of metaphase chromosomes by treatment authorities for providing all the facilities needed for the successful with hypotonic KCl. Stain Technol 1965;40(6):333-8. completion of this project. 12. Shukla Y, Taneja P. Antimutagenic effects of garlic extract on chromosomal aberrations. Cancer Lett 2002;176(1):31-6. 13. Bhattacharya K, Yadava S, Papp T, Schiffmann D, Rahman Q. Reduction REFERENCES of chrysotile asbestos-induced genotoxicity in human peripheral blood 1. Cavallito CJ, Bailey HJ. Allicin the antibacterial principle of Allium lymphocytes by garlic extract. Toxicol Lett 2004;153(3):327-32. sativum. I. isolation, physical properties and antibacterial action. J Am 14. Siddique YH, Afzal M. Antigenotoxic effect of allicin against methyl Chem Soc 1944;66(11):1950-1. methanesulphonate induced genotoxic damage. J Environ Biol 2. Budavari S, editor. The Merck Index, 12th ed., Version 12:3. Whitehouse 2005;26(3):547-50. Station, NJ, Boca Raton, FL: Merck & Co., Chapman & Hall/CRC 15. Dutta D, Devi SS, Krishnamurthi K, Kumar K, Vyas P, Muthal PL, et al. [CD-ROM]; 2000. Modulatory effect of distillate of Ocimum sanctum leaf extract (Tulsi) 3. Miron T, Shin I, Feigenblat G, Weiner L, Mirelman D, Wilchek M, on human lymphocytes against genotoxicants. Biomed Environ Sci et al. A spectrophotometric assay for allicin, alliin, and alliinase (alliin 2007;20(3):226-34. lyase) with a chromogenic thiol: Reaction of 4-mercaptopyridine with 16. Ghani MA. Determination of alliin and allicin in different types garlic thiosulfinates. Anal Biochem 2002;307(1):76-83. using high performance liquid chromatography. J Univ Anbar Pure Sci 4. Lawson LD, Wang ZJ. Low allicin release from garlic supplements: A 2010;4(2):16-23. major problem due to the sensitivities of alliinase activity. J Agric Food 17. Iberl B, Winkler G, Müller B, Knobloch K. Quantitative determination of Chem 2001;49(5):2592-9. allicin and alliin from garlic by HPLC*. Planta Med 1990;56(3):320-6. 5. Cruz-Villalon G. Synthesis of allicin and purification by solid-phase 18. Amagase H, Petesch BL, Matsuura H, Kasuga S, Itakura Y. Intake of extraction. Anal Biochem 2001;290(2):376-8. garlic and its bioactive components. J Nutr 2001;131(3s):955S-62.

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